Methods for the controlled delivery of pharmacologically active compounds

ABSTRACT

The present invention provides compositions and methods for extending the release times and lowering the toxicity of pharmacologically active compounds. The compounds comprise a salt of the pharmacologically active compound with a lipophilic counterion and a pharmaceutically acceptable water soluble solvent combined together to form an injectable composition. The lipophilic counterion may be a saturated or unsaturated C 8 -C 22  fatty acid, and preferably may be a saturated or unsaturated C 10 -C 18  fatty acid. The compounds precipitate in aqueous environments. When injected into a mammal, at least a portion of the composition precipitates and releases the active compound over time. Thus, the composition forms a slowly releasing drug depot of the active compound in the mammal. Therefore, the present invention enables one to provide a controlled dose administration of the active compound for a period of up to 15 days or even longer. Many compounds can be administered according to the present invention including, but not limited to, tilmicosin, oxytetracycline, metoprolol, fluoxetine, roxithromycin, and turbinafine.

This application is a continuation-in-part of U.S. application Ser. No.10/274,445, filed Oct. 18, 2002, which claims the benefit of U.S.application Ser. No. 60/343,625, filed Oct. 19, 2001, both of which arehereby incorporated by reference in their entireties including alltables, figures, and claims.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for extendingthe release times and decreasing the toxicity of pharmacologicallyactive compounds.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

It is often desirable to extend the release time of an injected drug toincrease its duration of action, or to reduce its toxic effects.Formulations that are readily soluble in the body are usually absorbedrapidly and provide a sudden burst of available drug as opposed to amore desirable and gradual release of the pharmacologically activeproduct. A variety of attempts have been made to provide controlled andextended release pharmaceutical compounds, but have not succeeded inovercoming all of the problems associated with the technology, such asachieving an extended release time, maximum stability and efficacy,reduced toxicity, maximum reproducibility in preparation, and theelimination of unwanted physical, biochemical, or toxicological effectsintroduced by undesirable matrix materials.

Oxytetracycline is a widely used and useful antibiotic for treatingvarious infections in mammals. In particular it is used for treating andpreventing respiratory infections in domestic animals. There aresignificant costs associated with repeated administrations throughconventional means.

Tilmicosin is a macrolide antibiotic with two tertiary amines. It has along tissue half-life and is effective against a broad range of bacteriaand is used to treat respiratory diseases in cattle. At elevated levelstilmicosin is cardiotoxic and its use in sensitive species such as cats,goats, pigs and horses has been avoided almost entirely due to safetyreasons. The commercial product, Micotil® (Eli Lilly & Co.,Indianapolis, Ind.), is a solution of the di-phosphate salt and isdescribed in U.S. Pat. No. 5,574,020. This formulation is effective incattle, but the antibiotic is released rapidly and results in toxicityin many species, including dogs and cats.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compositions for theadministration of pharmacologically active compounds to mammals. Thecompositions comprise a salt of a pharmacologically active compound witha lipophilic counterion, and a pharmaceutically acceptable water solublesolvent combined together under conditions to form an injectablecomposition. In one embodiment the composition precipitates wheninjected into water. The composition releases the pharmacologicallyactive compound over time when injected into a mammal. In variousembodiments, the compositions of the present invention include a widevariety of pharmacologically active compounds such as tilmicosin,oxytetracycline, doxycycline, metoprolol, sulfamethazine, trimethoprim,neomycin, streptomycin, gentamycin, dibucaine, bupivacaine, benzocaine,tetracaine, acepromazine, itraconazole, tetracyclines, sulfonamides,aminoglycosides, fluoxetine, roxithromycin, terbinafine, or anypharmacologically active compound with appropriate solubility andchemical functionalities. The lipophilic counterion may be a saturatedor unsaturated fatty acid of any specific number of carbons between 8and 22, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or22 carbons. In one embodiment the fatty acid is a C₈-C₁₈ fatty acid, andin another embodiment a C₁₀-C₁₈ fatty acid, such as lauric acid,linoleic acid, decanoic acid, myristic acid, or oleic acid. Otherlipophilic counterions may also be used, for example dicarboxylic acidssuch as sebacic acid, polymeric acids such as lipophilic poly-carboxylicacids, and aromatic acids, such as benzoic acid. The pharmaceuticallyacceptable carrier may be an organic solvent. In various embodiments,the solvent may be pyrrolidone, N-methyl pyrrolidone, polyethyleneglycol, propylene glycol (e.g., at about 10% in glycerol formal with orwithout stabilizers), glycerol formal, isosorbide dimethyl ether,ethanol, dimethyl sulfoxide, tetrahydrofurfuryl alcohol, triacetin, orany combination of these in any combined proportions, or another solventfound to have similar acceptable properties such as being non-toxic andsoluble in water.

In another embodiment the compositions of the invention are salts ofpharmacologically active compounds with a polycarboxylic acid counterionand a pharmaceutically acceptable water soluble solvent, combinedtogether under conditions to form an injectable composition thatprecipitates when injected into water at room temperature orprecipitates in physiological (“in vivo”) environments. The compositionsform a slowly releasing drug depot of the active compound and releasethe active compound over time when injected into a mammal. By“polycarboxylic acid” is meant a molecule containing at least twocarboxyl groups. In various embodiments the polycarboxylic acid ispolyaspartic acid, polyacrylic acid, sebacic acid, dodecanedioic acid,polysebacic acid, polybenzoic acid, or combinations thereof. By “poly”is meant two or more.

In one embodiment, the pharmacologically active compound isoxytetracycline, the lipophilic counterion is lauric acid, and thepharmaceutically acceptable solvent is propylene glycol, polyethyleneglycol, glycerol formal, or a combination of these. In anotherembodiment the pharmacologically active compound is tilmicosin, thelipophilic counterion is lauric acid, and the pharmaceuticallyacceptable solvent is propylene glycol, polyethylene glycol, glycerolformal, or a combination of these. In still another embodiment, thecompositions precipitate and release the active compound over time whenintroduced or injected into an aqueous environment. The compositions mayalso form a drug depot in the mammal when injected, which releases thecompound over time. As used herein, “about” means plus or minus 10%.

In another aspect the present invention provides a composition of alauric acid or decanoic acid salt of tilmicosin and a pharmaceuticallyacceptable solvent. At least a portion of the lauric acid or decanoicacid salt of tilmicosin is dissolved in the solvent, and the compositionprecipitates in water. In one embodiment the pharmaceutically acceptablesolvent comprises about 90% glycerol formal and about 10% propyleneglycol. In another embodiment the pharmaceutically acceptable solventcomprises N-methyl pyrrolidone. In one embodiment the lauric acid ordecanoic acid is present at about 2 molar equivalents to the tilmicosin,and in other embodiments is present at from about 1.5 molar equivalentsto 2.5 molar equivalents or more. By “di(lauric)” acid salt is meant twolauric acid molecules bound to the tilmicosin. By “at least a portion”is meant at least 10%. In various other embodiments “at least a portion”means at least 25% or 35% or 50% or 65% or 75% or 90% of the saltdissolves in the solvent and precipitates in water. A salt “dissolves”in the solvent when no more than 10% of the salt is retained on a 0.22um filter when the sample is mixed with the solvent and filtered at 98°F.±2° F. A composition “precipitates” in water when at least 10% of thecomposition is retained on a 0.22 um filter after being mixed with waterat 98° F.±2° F.

The present invention also provides novel methods of administeringcompositions and formulations of the present invention to mammals. Themethods involve administering active compounds that, if presented inpreviously available forms, may result in toxicity to the treatedmammal. Thus, the formulations and methods of the present inventionenable one to administer compounds that previously have not been able tobe widely used in particular mammals due to safety considerations. Themethods also enable one to extend the release times of compounds andprovide a controlled dose of active compound to the treated patient. Themethods of the present invention enable one to administer thepharmacologically active compound to the treated mammal in apharmaceutically effective amount for 4-15 days, such as for 1 day, 2days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days or 15 days, or even more. The precisetime will depend on several variables that may be manipulated tooptimize the present invention for a particular pharmacologically activecompound or application. In various embodiment the compound is presentin the treated tissue at a pharmaceutically effective amount for thetime periods described above after injection. In one embodiment apharmaceutically effective amount is present in the tissue for thenumber of days indicated after a single injection.

In another aspect, the present invention provides methods ofadministering a pharmacologically active compound to a mammal. Themethods comprise preparing a composition of the present invention, andinjecting the composition into the mammal. At least a portion of thecomposition precipitates and releases the pharmacologically activecompound over time when injected into the mammal.

In another aspect, the present invention provides methods of extendingthe release time and lowering the toxicity of a pharmacologically activecompound administered to a mammal. The methods comprise preparing aformulation of the invention, and injecting the composition into themammal. At least a portion of the composition precipitates and releasesthe pharmacologically active compound over time after injection into themammal, thereby extending the release time of the compound. Theinvention may therefore provide a controlled dosage of active compoundto the treated mammal. The present invention enables one to provide acontrolled dose administration of the active compound for periods of4-15 days or even longer, as described above.

In yet another aspect, the present invention provides methods ofmanufacturing an injectable formulation for the administration of apharmacologically active compound to a mammal. The methods includeforming a salt of the pharmacologically active compound with alipophilic counterion, providing a water soluble pharmaceuticallyacceptable solvent, combining the salt and the solvent under conditionsto form an injectable formulation, wherein at least a portion of theformulation precipitates and releases the pharmacologically activecompound over time when injected into the mammal. These methods can beused to manufacture the compositions

In another aspect the present invention provides compositions for theadministration of a pharmacologically active compound to a mammal. Thecompositions contain a salt of the pharmacologically active compoundwith a lipophilic counterion and a pharmaceutically acceptable solvent,combined together to form an injectable composition. At least a portionof the pharmaceutically active compound with lipophilic counteriondissolved in the solvent precipitates in vivo and releases the activecompound over time when injected into the mammal. The present inventiontherefore offers important advantages over formulations previouslyavailable. The present invention allows for the controlled release ofpharmacologically active compounds to reduce toxicity, particularly insmall animals such as dogs and cats. It also offers the advantage ofbeing able to administer compounds to domestic animals in an efficientmanner, thereby requiring a smaller investment in time and resourcesthan is available with previous modes of drug administration. Thepharmacologically active compound is available in a stable, injectable,formulation that precipitates when injected and slowly releases theactive compound over an extended period of time.

In another aspect the invention provides methods of treating mammals.The methods involve injecting a composition of the invention into themammal. In one embodiment the composition has a tilmicosin concentrationof from about 100 mg/ml to about 600 mg/ml and is injected at a dose offrom about 10 mg tilmicosin/kg to about 45 mg/tilmicosin/kg of mammal.In one embodiment the formulation has a tilmicosin concentration ofabout 300 mg/ml. In various embodiments, the composition has atilmicosin concentration of about 200 mg/ml, 300 mg/ml, 400 mg/ml, or500 mg/ml. In still other embodiments the dose is about 20 mg/kg, 30mg/kg, or 40 mg/kg. The mammal, may be a dog or a cat, such as thecommon house cat, Felis catus.

In another embodiment the invention provides methods for treatingmammals in need of such treatment. The methods involve administering toa mammal a composition of the invention according to the methodsdescribed herein. In one embodiment the composition is a lauric ordecanoic acid salt of tilmicosin and a pharmaceutically acceptablesolvent. The methods can be used to treat various diseases andconditions, such as Lyme disease, pustular contagious dermatitis, softtissue infections, an ear infection, and a urinary tract infection. Lymedisease occurs in humans, dogs, cats, horses, and cattle. Lyme diseaseis caused by the bacterial spirochete Borrelia (e.g., Borreliaburgdorferi). Pustular contagious dermatitis (also called “orf”) is acutaneous lesion caused by a parapox virus that occurs in sheep.Clinical features include one or more red lesions that develop on theudder and teats of the sheep. Soft tissue infections are most oftencaused by Staphylococcus aureus (Gram-positive cocci in clusters) andStreptococcus pyogenes (Gram-positive cocci in chains).

In another aspect the present invention provides methods of treatingrespiratory disease in a mammal. The methods involve administering tothe mammal a composition of the present invention according to themethods described herein. In various embodiments the respiratory diseaseis caused by the organism Bordetella bronchiseptica. This organism is aGram-negative, aerobic coccobacillus and is a primary pathogenresponsible for respiratory disease in many animal species. The organisminhabits the upper respiratory tract, but is associated with both upperand lower airway disease. The organism is responsible for causingvarious respiratory diseases. For example, in dogs the organism causeskennel cough (tracheobronchitis), rhinitis, and sinitus. In cats theorganism causes tracheitis, suppurative bronchopneumonia, andlymphadenitis. In pigs the organism causes atrophic rhinitis andpneumonia in pigs, and also causes respiratory diseases in rabbits suchas “snuffles” and serous to purulent rhinitis. In guinea pigs theorganism causes serous to purulent otitis media, necrotizing tracheitis,suppurative necrotizing bronchopneumonia. In rats the organism causesacute to subacute bronchopneumonia and atrophic rhinitis, as well ascausing respiratory diseases in primates. The organism is transferred bydirect animal to animal contact, as well as by airborne transmission.The terms of diseases described above are intended according to theirstandard clinical definitions.

In other embodiments the compositions and methods of the invention areuseful for treating respiratory diseases in mammals associated withmycoplasma. For example, mycoplasma hypopneumoniae is an obligatorypathogen of the respiratory tracts of pigs and rodents. The organismsinfects the airways of the respiratory system and is recognized as aninitiator or potentiator of more serious respiratory diseases caused byother primary or opportunistic pathogens. By “associated with”mycoplasma means that the presence of mycoplasma is an initiator orpotentiator of the disease.

The summary of the invention described above is not limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the preferred embodiments, as well asfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical illustration showing that oxytetracycline preparedaccording to the present invention is released into saline at a rateslower than that of the free drug. The solvent is DMSO, and thelipophilic counterion is lauric acid.

FIG. 2 is a graphical illustration showing that metoprolol preparedaccording to the present invention is released into saline at a rateslower than that of the free drug. Fatty acid and solvent are lauricacid and N-methylpyrrolidone.

FIG. 3 is a graphical depiction illustrating that the rate of release ofthe pharmacologically active compound (tilmicosin) is affected by thechain length of the fatty acid selected. Solvent: N-methyl pyrrolidone;lipophilic counterion: decanoic acid and lauric acid.

FIG. 4 is a graphical depiction illustrating the solvent effect on invitro release kinetics in tilmicosin. Lipophilic counterion:di(decanoic) acid; tilmicosin at 100 mg/ml in the formulation.Abbreviations are as follows: PEG=polyethylene glycol,THFA=tetrahydrofurfuryl alcohol, DMA=dimethyl acetamide,ISO-DME=isosorbid dimethyl ether, DMSO=dimethyl sulfoxide, NMP=N-methylpyrrolidone.

FIG. 5 is a graphical depiction illustrating that the rate of release ofthe pharmacologically active compound (tilmicosin) is a function of theconcentration of fatty acid salt. Lipophilic counterion: decanoic acid.

FIG. 6 is a graphical depiction illustrating the in vitro releasekinetics of fluoxetine:lauric acid fatty acid salt formulation.

FIG. 7 is a graphical depiction of the pharmacokinetics of fluoxetinehydrochloride (HCl) and fluoxetine:lauric acid fatty acid salt (FAS) incats.

FIG. 8 is a semilog plot of tilmicosin concentration in cat lung tissueover 21 days. Eight male and eight female cats were used and dosed with10 mg/kg of body weight for all tissue types.

FIG. 9 is a semilog plot of tilmicosin concentration in cat kidneytissue over 21 days. Eight male and eight female cats were used anddosed with 10 mg/kg of body weight for all tissue types.

FIG. 10 is a semilog plot of tilmicosin concentration in cat livertissue over 21 days. Eight male and eight female cats were used anddosed with 10 mg/kg of body weight for all tissue types.

DETAILED DESCRIPTION OF THE INVENTION

The compositions of the present invention may be prepared using salts ofpharmacologically active compounds with basic functionalities. These canbe made using a variety of lipophilic acids, saturated or unsaturatedfatty acids, cholic acids, phosphatidic acids, dicarboxylic acids suchas sebacic acid or any acid that, when combined with thepharmacologically active compound, renders the resulting salt insolublein water, but soluble in a water soluble solvent. By “salt” is meant twocompounds that are not covalently linked but are chemically boundthrough ionic attractions. In one embodiment the formulations of thepresent invention are non-aqueous. The bond in a salt may be a result ofa combination of an ionic bond and a hydrogen bond. Thus, for example, a“lauric acid salt” of tilmicosin refers to tilmicosin bound to lauricacid through an ionic attraction. By “water miscible” is meant that thesolvent is capable of mixing in any ratio in water without separation oftwo phases. By “water soluble” is meant that the solvent has at leastsome significant level of solubility in aqueous solutions, e.g.,triacetin is considered a water soluble solvent since it is soluble inwater at a ratio of about 1:14. By a “lipophilic counterion” is meant anionic form of a fat soluble molecule. The lipophilic counterion may be afatty acid, but may also be another fat soluble molecule. The counterionhas at least one charge opposite to that of a chemical group on anopposing salt member, thereby causing an ionic attraction between thetwo molecules. The particular water/octanol partition coefficient of alipophilic counterion will vary. In one embodiment the lipophiliccounterions have a water/octanol partition coefficient of 100 orgreater. In other embodiments the coefficient is 50 or greater (e.g.,benzoic acid), or 40 or greater, or 25 or greater, or 10 or greater. By“injectable formulation” or “injectable composition” is meant aformulation or composition that can be injected, i.e., drawn into asyringe and injected subcutaneously, intraperitoneally, orintra-muscularly muscularly into a mammal without causing adverseeffects due to the presence of solid materials in the composition. Solidmaterials include, but are not limited to, crystals, a gummy mass, and agel. By “pharmacologically active compound” is meant a chemical compoundthat causes a pharmacological effect in the treated mammal. For example,the effect may be to destroy, hinder, or prevent growth of bacteria orparasites, reduce inflammation, or another pharmaceutical and measurableeffect in the treated mammal.

By the verb “precipitate” is meant that the compound forms aprecipitate, or solid. A precipitate is an insoluble solid formed insolution at room temperature in vitro or in a physiological (in vivo)environment. The precipitate can take many forms such as, for example, asolid, a crystal, a gummy mass, or a gel. By “pharmaceutically effectiveamount” is meant an amount that exerts a measurable and medicallysignificant effect on the treated mammal, resulting in progress towardscuring, arresting, or preventing the subject disease, or alleviating orpreventing the condition that was the reason for treatment. A“pharmaceutically acceptable solvent” is a liquid that dissolves a saltof the pharmacologically active compound and a lipophilic counterion,and that is suitable for use with humans and/or animals without undueadverse side effects (such as toxicity, irritation, and allergicresponse) and commensurate with a reasonable benefit/risk ratio.

The compositions of the present invention offer several advantages. Thecompositions are injectable compositions that contain highconcentrations of the active compound. In various embodiments, thepharmacologically active compound may be loaded into the composition inthe range of 10%-60% (w/v). But the person of ordinary skill in the artwill realize this range may be varied widely, depending on thesolubility or insolubility of the pharmacologically active compound, thelipophilic counterion selected, the solvent selected, the injectabilityof the final product, and any other relevant needs of the particularapplication. For example, pharmacologically active compound can beloaded at a concentration of at least 20%, 30%, 40%, or 50% (w/v), andmay also be loaded as low as 10%, or 5%, or even 1% and still provide auseful effect. Similarly, active compound may be loaded at 70%, 80%, oreven higher as needs require. No exotic excipients or carriers arerequired. The compositions are easily filtered, thereby simplifying themanufacturing process. It is believed that the exclusion of water fromthe formulation should impart greater stability to the formulation, andinhibit the growth of microorganisms. The processes for preparing thecompositions, as described herein, are simple, and administrationaccording to the present invention results in milder reactions at theinjection site due to the neutralization of the pharmacologically activecompound.

The present invention provides the ability to modulate the release rateand release time of the pharmacologically active compound. The releaserate may be modulated by varying the lipophilicity and molecular weightof the counter-ion used to make the salt. For example, lauric acid saltsof tilmicosin are usually released more slowly than decanoate salts. Inaddition, higher concentrations of the salt in the formulation usuallyyield slower release rates. For example, the decanoate salt oftilmicosin is released more slowly from a 60% tilmicosin-fatty acid saltformulation that from a 30% tilmicosin-fatty acid salt formulation.Similarly, as explained herein, other variables such as selection oflipophilic counterion, solvent selection, salt concentration, and othersmay be manipulated to lengthen or shorten the release time of the activecompound to the desired point. Generally, it may be desirable for saltsto be based on the molar ratio of charged groups. But one may alsosuccessfully create insoluble salts by utilizing a hemi-salt or byotherwise varying from a 1:1 ratio. The pharmaceutically acceptablesolvent may be a water miscible or water soluble solvent. Mixtures ofwater soluble and/or water miscible solvents may also be utilized. Theperson of ordinary skill in the art will realize that various watersoluble solvents may be mixed to optimize the result for a particularapplication. For example, a mixture of polyethylene glycol, propyleneglycol, and glycerol formal may be mixed in various ratios to provide anoptimal solvent. In some embodiments, mixing in approximately evenamounts may provide a suitable solvent.

In other embodiments, formulations of the invention containing a salt ofthe pharmacologically active compound with a lipophilic counterion canbe combined with the unsalted form of the active, in order to provide agreater initial dose of active compound.

Without wanting to be bound by any particular theory, injectablecompositions may be obtained when a salt is formed of apharmacologically active agent with a lipophilic counterion, andcombined with a parenteral organic solvent. It is believed that whenthis formulation is injected into a mammal, the solvent may diffuse awayfrom the injection site as aqueous body fluids diffuse towards the site,resulting in the precipitation of the pharmacologically active compoundin the treated mammal. The precipitate may take many forms, for example,a solid, crystals, a gummy mass, or a gel. There will thus exist aconcentration of the active compound that is released in apharmaceutically effective amount over a desired period of time. Theprecipitate may act as a drug depot in the mammal resulting in therelease of the compound over a period of time. Release times may beobtained of at least 3 days, at least 4 days, at least 5 days, at least6 days at least 7 days, or any specific number of days up to andincluding at least 15 days, or even longer, as desired. By “drug depot”is meant a concentration or precipitation of pharmacologically activecompound within the body of the treated mammal that releases apharmaceutically effective amount of the active compound over time.

It was shown that the fatty acid chain length, the particularcombinations of fatty acids, the percent pharmacologically activecompound:lipophilic counterion salt in the formulation, and thepharmaceutically acceptable solvent selected all influence the releasekinetics of the pharmacologically active compound. Thus, the releasekinetics of the pharmacologically active compound may be convenientlyand easily managed by manipulating these and other variables. It wasalso found that the formulations retained stability during sterilizationby autoclave. The present invention may be applied to manypharmacologically active compounds that have an appropriate solubilityand chemical functionality. Thus, it is contemplated that the presentinvention may be applicable to a wide variety of pharmacologicallyactive compounds, such as drugs, medications, nutrients, or otherdesirable compounds for administration to a mammal.

Some modifications to the methods presented herein may be desirablebased on the particular characteristics of the pharmacologically activecompound involved. The following non-limiting examples present furtherapplications of the present invention and are provided by way of exampleonly.

EXAMPLE 1 Oxytetracycline

Oxytetracycline has one tertiary amine group, and the hydrochloride saltof oxytetracycline is readily soluble in water. We have found thatadding one mole of fatty acid to one mole of oxytetracycline creates asalt that has lower solubility in water but is more soluble thanunmodified oxytetracycline in N-methylpyrrolidone (NMP). When water isadded to the NMP formulation, the salt precipitates.

The in vitro rate of release of oxytetracycline may be determined bysealing the formulation in a dialysis bag (Pierce, Rockford, Ill.),placing it in a reservoir of saline solution, and measuring the amountof drug in the saline as a function of time. The formulation of thepresent invention was compared with other oxytetracycline formulations.FIG. 1 shows that the oxytetracycline formulation of the presentinvention is released into the saline at a rate substantially slowerthan that of the free drug.

An oxytetracycline composition according to the present invention wasprepared by adding 0.464 grams of oxytetracycline and 0.203 grams oflauric acid to 3 ml of NMP. The mixture was stirred for 60 minutes,resulting in a clear solution. 1 ml of this solution was sealed in adialysis bag, and the bag was suspended in 150 ml of phosphate-bufferedsaline, pH 7.4. Aliquots were removed at various intervals andoxytetracycline concentration was determined by spectrophotometry. Theresults in FIG. 1 show that oxytetracycline continued to diffuse out ofthe bag for more than 120 hours, at which point only about 50% of theoxytetracycline present had been released.

EXAMPLE 2 Metoprolol

Metoprolol is an antihypertensive, antianginal and antiarrhythmic drug,of the following structure:

Its succinate and tartarate salts are available commercially underseveral trade names. Both these salts as well as the base form of thedrug are highly soluble in water. The base form of the metoprolol wasprepared from commercially available tartarate salt by standardprocedure. When the amine group of metoprolol is neutralized with lauricacid, the resulting salt is poorly soluble in water, but readily solublein pharmaceutically-acceptable non-aqueous solvents. A metoprololcomposition according to the present invention were prepared by adding0.3224 grams of metoprolol base and 0.2661 grams of lauric acid to 2.415ml of NMP. The mixture was stirred for 30 minutes, resulting in a clearsolution. One ml of this solution was sealed in a dialysis bag, and thebag was suspended in 150 ml of phosphate-buffered saline, pH 7.4.Aliquots were removed at various intervals and metoprolol concentrationwas determined by spectrophotometry. The results in FIG. 2 show thatmetoprolol continued to diffuse out of the bag for more than 48 hourswhile the control solution of metoprolol base (prepared by dissolving150 mg in 1.124 ml of NMP) is diffused off rapidly.

EXAMPLE 2A Tilmicosin

Tilmicosin is an antibiotic in the macrolide class with the followingstructure:

It is effective against a broad range of bacteria, and is used for thetreatment of respiratory diseases in cattle. The basic form ismoderately soluble in aqueous solutions, while the chloride andphosphate salts are highly soluble. At elevated levels, tilmicosin iscardiotoxic, and therefore is not administered intravenously. For safetyreasons, its use has been avoided almost entirely in sensitive speciessuch as cats, goats, pigs, and horses.

When the two amine groups of tilmicosin are neutralized with any ofseveral fatty acids (such as, for example, decanoic C₁₀, lauric C₁₂,myristic C₁₄, palmitic C₁₆, stearic C₁₈, oleic C₁₈, elaidic C₁₈,linoleic C₁₈, and erucic C₂₂, sebacic, dodecanedioic), the resultingsalt is poorly soluble in water, but readily soluble inpharmaceutically-acceptable non-aqueous solvents. When the formulationof the salt is sealed in a dialysis cassette and place in saline, thetilmicosin salt precipitates, and tilmicosin is slowly released from thebag. The rate of release is a function of the chain length of the fattyacid (FIG. 3), the solvent (FIG. 4) and the tilmicosin-fatty acid saltconcentration (FIG. 5).

EXAMPLE 3 Tilmicosin Salts In Vitro

10 grams (0.0115 moles) of tilmicosin and 0.0253 moles of variouscarboxylic acids (such as, for example, decanoic, lauric, linoleic, ormyristic acids, in individual assays) were taken in a flask and made upto a final volume of 100 ml with N-methyl-pyrrolidone and stirred for 60minutes to obtain a clear solution. One ml aliquots of these solutionswere sealed in dialysis bags, and the bags were suspended in flaskscontaining 150 ml of phosphate-buffered saline, pH 7.4. The salt wasobserved to precipitate in the bag within about 1 hour. Aliquots ofsaline were removed at various intervals and tilmicosin was determinedby HPLC. The results with decanoic acid (C-10) and lauric acid (C-12)salts in FIG. 3 show that tilmicosin continued to diffuse out of the bagfor more than 120 hours. Longer chain length acids caused a slowerrelease of tilmicosin. Micotil® (Eli Lilly, Indianapolis, Ind.), aphosphate salt of tilmicosin, is readily soluble and rapidly diffusesfrom the bag.

EXAMPLE 4 Tilmicosin-di(Decanoic Acid) in Various Solvents

Solutions of tilmicosin di(decanoic acid) salt were prepared in varioussolvents, combining 10 grams (0.0115 moles) of tilmicosin and 0.0253moles of decanoic acid in various solvents to a final volume of 100 ml.The in vitro release rates were measured using the dialysis method ofExample 1. The results in FIG. 4 show that the release rate varies withthe solvent, but that all of the solvents yield a slower release ratethan that observed with the phosphate salt (Micotil (R)) shown in FIG.3.

EXAMPLE 5 Tilmicosin Release Concentration Effect

Solutions of tilmicosin: di(decanoic acid) salt were prepared bycombining 30 grams (0.0345 moles) or 60 grams (0.0690 moles) oftilmicosin with 2 equivalents of decanoic acid in NMP to a final volumeof 100 ml. The in vitro release rates were measured using the dialysismethod of Example 1, and the data in FIG. 5 show that higher startingconcentrations result in a slower release rate.

EXAMPLE 6 Tilmicosin In Vivo

Formulations of tilmicosin decanoate, laurate, and myristate (i.e., withtwo equivalents of the respective acid) were formulated at 100 mg/ml inN-methyl pyrrolidone and subcutaneously injected into cats in the backof the neck at a dosage of 45 or 75 mg/kg of body weight. Previous dataindicates that a dosage of 25 mg/kg of the phosphate salt of tilmicosincould be fatal to cats. The cats showed hypothermia and lethargy afterinjection, indicative of the bioavailability of the drug. The toxicitywas found to be substantially less in formulations with fatty acid chainlengths greater than C₁₀, consistent with the slower release of drugfrom these formulations. All of the cats survived and were behavingnormally 3 days after the injection. The results are summarized in thefollowing Table 1.

Table 1: Blood levels of Tilmicosin at Specific Time Intervals

100 mg/ml of a formulation of a salt of tilmicosin with decanoic acid,lauric acid, or myristic acid in N-methyl pyrrolidone were injected into9 healthy adult cats at the dosages indicated. The resultingconcentrations in blood cells and plasma for each individual cat at 6hours and 2 days are shown in Table 1.

TABLE 1 6 hrs 6 hrs Day2 Day2 Cat Fatty Blood cells Plasma Blood cellsPlasma No. Acid Dosage (μg/ml) (μg/ml) (μg/ml) (μg/ml) 1 C-12 45 mg/kg5.8 11.3 0.9 0.5 2 C-12 75 mg/kg 5.5 18.0 1.5 1.5 3 C-12 75 mg/kg 2.59.9 0.9 1.0 4 C-14 45 mg/kg 2.4 7.5 0.9 1.1 5 C-14 75 mg/kg 6.7 11.6 9.14.6 6 C-14 75 mg/kg 3.5 6.1 1.5 0.7 7 C-10 45 mg/kg 3.7 15.9 8 C-10 75mg/kg 4.2 18.4 9 C-10 45 mg/kg 3.3 9.1

Tilmicosin salts were also studied in tissue. A tilmicosin:dilauricfatty acid salt in 10% propylene glycol in glycerol formal at 100 mg/mlwas administered subcutaneously at 10 mg/kg dose and the biodistributionin cats was determined.

The methods described here were developed for the determination andquantification of tilmicosin in various animal tissues and serum,particularly feline liver, kidney and lung tissue and serum. Withreference to the present disclosure it will be understood that manyvariations of the methods described herein are possible withoutdeparting from the invention.

The kidney, liver and lung tissue samples were collected 2, 3, 4, 7, 14,and 21 days after injecting the animals with tilmicosin formulation at10 mg/kg of body weight. The results are presented in FIGS. 8, 9 & 10.It was found that kidney is the marker tissue in cats while the liver isthe marker in cows and pigs. The levels in kidney were consistentlyhigher than in liver with Cmax (kidney: 13.8 mcg/gm; Liver: 7.3 mcg/gm)reached around 48 hours in both tissues. The Cmax for lung was found tobe 7.5 mcg/gm and was observed around 48 hours after injecting the dose.Detectable levels of tilmicosin persisted in tissues through day 21 ofthe study.

A drug extraction efficiency of ˜98% was obtained at a concentration of1 mcg/gm of tissue. The limit of detection for tilmicosin in variousfeline tissues was determined to be 0.032 mcg/gm. For feline serum, adrug extraction efficiency of 95% was obtained over a concentrationrange of 0.15 to 6 mcg/ml after fortification. The limit of detectionwas determined to be 0.16 mcg/ml with linearity extending from 0.15 to 6mcg/ml.

Preparation of Tissue Samples

Tissue samples were prepared by mincing with scissors or a scalpel on apaper towel. 10 ml of methanol was added to each tube and the sampleshomogenized separately for 10 to 15 minutes. Samples were sonicated onice for one minute and centrifuged at 10,000 rpm for 30 minutes at 4 C.The methanol extract was decanted into fresh centrifuge tubes and thetissue samples resuspend in the tubes with 10 mL of methanol and 5.0 mLof 100 mM phosphate. The tubes were vortexed and centrifuged at 10,000rpm for 30 minutes at 4 C. The extract was decanted into freshcentrifuge tubes and centrifuged at 5000 rpms for 10 minute at 4 C. Themethanol extract was added to 70 mL of water in a flask and swirled inthe flask to mix.

Each pool of extracts was loaded to a solid phase extraction C₁₈ Sep-PakPlus® (SPE) cartridge (Waters, Milford, Mass.) using a vacuum manifoldor hydrostatic pump to draw the pools through the cartridges. Oncesample were completely loaded, each SPE cartridge was washed with 10 mLof water, and then with 10 mL of 25% acetonitrile/water at a flow rateof less than 5 mL/minute. The SPE cartridges were disconnected from theapparatus and dried completely under high vacuum (26 in. Hg) in a vacuumdesiccation jar for 10 minutes.

Analyte was eluted from the SPE cartridges with 5% acetic acid/methanol.Only the first 2.0 mL of eluate was collected. The volumetric flaskswere inverted and mixed, and stored overnight at 4 C.

The sample eluates were filtered through a 0.22 um PVDF filter, andanalyzed by HPLC on a SphereClone® 5 um phenyl column (Phenomenex,Torrance, Calif.).

EXAMPLE 7 Fluoxetine

Fluoxetine is a selective serotonin reuptake inhibitor and isextensively used to treat psychological disorders such asobsessive-compulsive disorder in humans. It is shown that fluoxetine iseffective for treating aggressive behavior and separation anxiety indogs and urine spraying behavior in cats. Fluoxetine is formulated at100 and 150 mg/ml as a lauric acid salt in 10% propylene glycol inglycerol formal in 1:1 drug to fatty acid ratio. In vitro releasekinetics were studied for both formulations at 100 mg/mL concentrationusing the dialysis technique described in Example 1, and the resultspresented in FIG. 6. Fluoxetine base in 10% propylene glycol in glycerolformal at 100 mg/ml is used as a control in the experiment. While thefluoxetine base formulation is released for about 160 hrs, the 1:1(drug:LA) lauric acid salt formulation was released for 700 hrs.

The fatty acid salt formulation with 1:1.1 fluoxetine to lauric acidratio at 150 mg/mL concentration was injected sub cutaneously into catsat 20 and 30 mg/kg. At the same time two cats were dosed at 1 mg/kg/dayorally for 28 days. Serum samples were collected through day 42 andanalyzed for fluoxetine by HPLC using 35% acetonitrile in phosphatebuffer, pH 2.7 on a C₁₈ column. The results suggest that a single subcutaneous injection of fatty acid salt formulation provide drugconcentrations comparable to daily oral dose through 42 days (FIG. 7).

The fluoxetine was also formulated as linoleic acid salt in 10%propylene glycol in glycerol formal, yielding a clear solution. Thisformulation was also found to precipitate in water.

EXAMPLE 8 Roxithromycin

Roxithromycin is an antibiotic in the macrolide class with the followingstructure:

It is effective against a broad range of bacteria, and is used for thetreatment of respiratory diseases in cattle. The amine group ofroxithromycin was neutralized with linoleic acid in 10% propylene inglycerol formal, resulting in a clear solution at 200 mg/ml. As in thecase of tilmicosin, the salt precipitated when the solution was spikedin water.

EXAMPLE 9 Terbinafine

Terbinafine is an antifungal and the structure is as shown below:

Terbinafine is a specific inhibitor of squaline epoxidase, a key enzymein fungal ergosterol biosynthesis. The amine group of terbinafine wasneutralized with linoleic acid in 10% propylene glycol in glycerolformal, resulting in a clear solution at 150 mg/ml. The resulting saltwas highly insoluble in water, and precipitated when spiked into water.

EXAMPLE 10 Tilmicosin-decanoic Acid Salt Tissue Levels in Dogs

This example illustrates the tissue concentration over time in dogstreated with a formulation of the present invention containingtilmicosin and decanoic acid. 250 g of tilmicosin was combined with 104g of decanoic acid, and 103.61 g of propylene glycol to arrive atcomposition containing 25% tilmicosin, ˜2.1 molar equivalents ofdecanoic acid, and 10% propylene glycol. An overhead stirrer was used tostir the composition and the weight was brought to 1,142.17 g withglycerol formal. The mixture was stirred continuously until a clear,homogenous solution was obtained.

The composition was administered subcutaneously to 12 dogs (6 hounds and6 beagles) at a dosage of 15 mg/kg. The dogs were sacrificed on day 2,4, and 10 and tissue samples collected from the lung, liver, and kidney.A tilmicosin extraction procedure was performed on the tissue and theextracted sample was analyzed for the presence of tilmicosin using HPLC.The data is illustrated in Table 2 below.

Dose Lung mean Liver mean Kidney mean (mg/ mcg/ mcg/ mcg/ mcg/ mcg/ mcg/Dog kg) gm gm gm gm gm gm Day Beagle 1 15 8.52 15.68 11.34 2.00 Beagle 215 10.23 21.68 11.47 2.00 Hound 1 15 7.54 14.00 9.72 2.00 Hound 2 152.73 5.02 4.73 2.00 7.26 14.1 9.32 Beagle 3 15 2.49 6.61 6.02 4.0 Beagle4 15 2.23 4.78 5.77 4.0 Hound 3 15 2.04 3.79 4.70 4.0 Hound 4 15 1.292.58 2.45 4.0 2.01 4.44 4.74 Beagle 5 15 0.31 1.16 0.40 10.0 Beagle 6 151.00 3.26 1.36 10.0 Hound 5 15 0.75 4.05 0.63 10.0 Hound 6 15 0.76 0.861.17 10.0 0.71 2.33 0.89

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are set forth within the following claims.

1. A method of treating a respiratory disease in a mammal comprising: administering to the mammal a composition comprising a salt of a pharmacologically active compound with a lipophilic counterion and a pharmaceutically acceptable solvent; wherein the salt of the pharmacologically active compound and lipophilic counterion are combined together to form an injectable composition that precipitates when injected into water; and wherein the composition releases the active compound over time when injected into the mammal.
 2. The method of claim 1 wherein the pharmaceutically acceptable solvent is a water miscible solvent.
 3. The method of claim 1 wherein the pharmacologically active compound is an antibiotic.
 4. The method of claim 1 wherein the pharmacologically active compound is tilmicosin, oxytetracycline, doxycycline, fluoxetine, roxithromycin, terbinafine or metoprolol.
 5. The method of claim 1 wherein the pharmacologically active compound is selected from the group consisting of: trimethoprim, neomycin, streptomycin, gentamycin, dibucaine, bupivacaine, benzocaine, tetracaine, acepromazine, itraconazole, tetracyclines, sulfonamides, and aminoglycosides.
 6. The method of claim 1 wherein the lipophilic counterion is a C₁₀-C₂₂ saturated or un-saturated fatty acid.
 7. The method of claim 1 wherein the lipophilic counterion is a C₁₀-C₁₈ saturated or unsaturated fatty acid.
 8. The method of claim 7 wherein the fatty acid is selected from the group consisting of: lauric acid, decanoic acid, myristic acid, oleic acid and linoleic acid.
 9. The method of claim 1 wherein the lipophilic counterion is a polycarboxylic acid.
 10. The method of claim 9 wherein the polycarboxylic acid is selected from the group consisting of: sebacic acid, polysebacic acid, dodecanedioic acid, polyaspartic acid, polyacrylic acid, and polybenzoic acid.
 11. The method of claim 1 wherein the pharmaceutically acceptable solvent is selected from the group consisting of one or a combination of: pyrrolidone, N-methyl pyrrolidone, polyethylene glycol, propylene glycol, glycerol formal, isosorbide dimethyl ether, ethanol, dimethyl sulfoxide, triacetin, and tetrahydrofurfuryl alcohol.
 12. The method of claim 1 wherein the pharmaceutically acceptable solvent comprises 10% propylene glycol in glycerol formal with or without stabilizers.
 13. The method of claim 1 wherein the mammal is treated for a respiratory disease associated with Mycoplasma.
 14. The method of claim 1 wherein the pharmacologically active compound is oxytetracycline, the lipophilic counterion is lauric acid, and the pharmaceutically acceptable solvent is selected from the group consisting of one or more of polyethylene glycol, propylene glycol, and glycerol formal.
 15. The method of claim 1 wherein the pharmacologically active compound is tilmicosin, the lipophilic counterion is lauric acid, and the pharmaceutically acceptable solvent is selected from the group consisting of one or more of polyethylene glycol, propylene glycol, and glycerol formal.
 16. The method of claim 1 wherein the respiratory disease is caused by a Bordetella.
 17. The method of claim 1 wherein the mammal is selected from the group consisting of a dog, a cat, a guinea pig, a pig, and a rodent.
 18. The method of claim 1 wherein the respiratory disease is kennel cough and the mammal is a dog or a cat.
 19. A method of treating a mammal in need thereof comprising: administering to the mammal a lauric acid or decanoic acid salt of tilmicosin and a pharmaceutically acceptable solvent; wherein the lauric acid or decanoic acid and tilmicosin are combined to form an injectable composition that precipitates when injected into water; and wherein the composition releases the active compound over time when injected into the mammal.
 20. The method of claim 19 wherein the pharmaceutically acceptable solvent comprises about 90% glycerol formal and about 10% propylene glycol.
 21. The method of claim 19 wherein the pharmaceutically acceptable solvent comprises N-methyl pyrrolidone.
 22. The method of claim 19 wherein the composition is administered by injection and has a tilmicosin concentration of about 100 mg/ml and is injected at a dose of about 10 mg tilmicosin/kg of mammal.
 23. The method according to claim 22 wherein the mammal is a dog or a cat.
 24. The method of claim 19 wherein the composition comprises a decanoic acid salt of tilmicosin and a pharmaceutically acceptable solvent; and wherein at least a portion of the decanoic acid salt of tilmicosin is dissolved in the solvent.
 25. The method of claim 24 wherein the pharmaceutically acceptable solvent comprises about 90% glycerol formal and about 10% propylene glycol.
 26. The method of claim 24 wherein the pharmaceutically acceptable solvent comprises N-methyl pyrrolidone.
 27. The method of claim 19 wherein the mammal is treated for a respiratory disease caused by a Bordetella.
 28. The method of claim 19 wherein the mammal is treated for a respiratory disease associated with Mycoplasma.
 29. The method of claim 19 wherein the mammal is treated for a disease selected from the group consisting of: Lyme disease, pustular dermatitis, a soft-tissue infection, an ear infection, and a urinary tract infection. 